Project Summary Significant progress has been made in defining the key cellular, molecular and physiologic determinants of the adult bone marrow (BM) hematopoietic stem cell (HSC) niche that regulate HSC function during steady-state hematopoiesis. Even with this increased knowledge, little remains known about the factors responsible for mediating recovery of HSC numbers following significant HSC loss in vivo. In this proposal, we utilize clonal, adherent primary BM mesenchymal stromal/stem cells (MSC) that uniformly express molecules that are hallmarks of adult MSC, to define critical factors regulating HSC self-renewal. Given the cellular and physiologic complexity of the BM niche, it is not possible to evaluate the inherent self-renewal-promoting potential of a given cell type by deleting that cell type in vivo as this only defines whether that cell type is necessary, through direct or indirect mechanisms, to maintain HSC survival, localization, and/or function within the niche. By using a highly defined in vitro co-culture system, we have shown that clonal primary MSC have a much higher intrinsic ability to promote HSC cell divisions leading to expansion (symmetric self-renewal) and that this ability is inversely correlated with the stage of osteoblast maturation such that mature osteoblasts have very limited intrinsic ability to support HSC self-renewal. This system is robust, with a 20-fold expansion of functional LT-HSC occurring after 10 days of co-culture and up to ~100-fold expansion occurring when Wnt signaling is blocked as measured by the gold-standard competitive repopulating cell assay. Although it could rightly be argued that any in vitro system represents a dramatic oversimplification of the in vivo HSC niche, the results from these studies will nevertheless provide a tractable model for delineating the essential components of Wnt signaling that are regulating HSC self-renewal using a clonal, primary BM cell type that is likely the closest functional analog to the prototypic perivascular stromal cell that maintains (and perhaps expands) HSC during homeostasis and under physiologic stress conditions. Understanding factors regulating LT-HSC expansion is vital for enhancing clinical applications like gene therapy, BM transplantation, and somatic cell gene correction of inherited blood disorders. It is also important for understanding basic molecular mechanisms regulating symmetric versus asymmetric stem cell divisions regulated by Wnt signaling. The overall hypothesis of this proposal is that altering the balance between canonical and non-canonical Wnt signaling functions to regulate whether LT-HSC self-renewal or differentiation occurs in the context of primary Nestin+Lepr+ BM MSC. This hypothesis will be addressed by: (1) determining the contributions of canonical and non-canonical Wnt signaling to promotion of LT-HSC self-renewal and differentiation in the context of primary BM-derived MSC clones, (2) biochemically purifying and functionally characterizing soluble Wnt ligands and other factors being inhibited by Wif1 in LT-HSC/MSC co-cultures, and (3) determining whether WNT regulation of LT-HSC self- renewal in human NESTIN+LEPR+ MSC and CD34+ cell co-cultures is conserved between mouse and man.